Mammals must take in nutrients from outside the body, and many transporter proteins (transporters) are known to exist in mammalian cells. These transporters mainly act to transport substances essential to the maintenance of life (amino acids, sugars, and such) into cells. In the physiological environment, cells often have multiple transporters that transport the same substrate. In these cases, the individual contribution of transporters to cellular uptake can be estimated using kinetic analysis (calculation of Km, Vmax, and so on; e.g., Wright E. M., Am. J. Physiol. Renal Physiol., 2001, 280: F10-18). Thus, identification of transport substrates and kinetic analysis of transporters are extremely important for revealing their physiological function and their potential in drug delivery.
Currently, methods for analysing transporter function use the following resources as materials: (1) primary cultured cells and cell membrane vesicles (such as lung cells and brush border membrane vesicles) comprising transporters isolated from living bodies; (2) cell lines derived from transporter-comprising cancer cells and so on (such as Caco-2 cells); (3) mammalian cells introduced with transporter genes (such as LLC-PK1 cells and MDCK cells) and Xenopus oocytes; and (4) insect cell membranes (such as Sf9 cell membranes) in which transporters have been expressed using baculovirus expression systems. Of these, mostly used are gene expression systems from mammalian cells and Xenopus oocyte cells. However, even in mammalian and Xenopus oocyte cells introduced with transporter genes, activities from endogenous transporters can be detected, thus elevating background levels (Kanai Y. et al., J. Clin. Invest. 93: 397-404 (1994); Kekuda R. et al., J. Biol. Chem. 271: 18657-18661 (1996); Kekuda R. et al., Am. J. Physiol. 272: G1463-1472 (1997); Yabuuchi H. et al., J. Pharmacol. Exp. Ther. 286: 1391-1396 (1998); Hatanaka T. et al., J. Clin. Invest. 107: 1035-1043 (2001)). For this reason, in some types of transporters, there are reports that describe an activity ratio of only two between cells introduced with genes and those not introduced with genes (parent cell lines). Carrying out kinetic analysis can be problematic in such gene-introduced cells with a low activity ratio.
In Xenopus oocyte cells introduced with transporter genes, transporter activity can be measured using electrophysiological methods. In transporters driven by Na and H ions, and substrates having an electric charge at physiological pH, transporter activity can be detected by measuring the electrical current caused by substrate transport. However, measuring transport activity is difficult when there is no driving force and also when substrates are electrically neutral at physiological pH. Kinetic analysis is also difficult in cases where transporter activity is observed but only a weak current can be detected. In addition, since electrophysiological methods require specific equipment, they are not simple or convenient.
The activity and substrate specificity of transporters that transfer drugs into cells has been reported to influence the drug's bioavailability (for example, Ganaphthy, Leibach, Curr. Biol 3: 695-701 (1991); Nakashima et al., Biochem. Pharm. 33: 3345-3352 (1984); Friedman, Amidon, Pharm. Res. 6:1043-1047 (1989); Okano et al., J. Biol. Chem. 261: 14130-14134 (1986); Muranushi et al., Pharm. Res. 6: 308-312 (1989); Friedman, Amidon, J. Control. Res. 13: 141-146 (1990)). In recent years, research on factors that fluctuate in vivo pharmacokinetics has clarified that drug-metabolising enzymes as well as drug-transporters influence the function of drugs in the body. Known drug-transporters include p-glycoprotein (Annu. Rev. Biochem. 58: 137 (1989)), multidrug resistance protein (Science 258: 1650 (1992); Cancer Res. 55: 102 (1995)), lung resistance protein (Ann. Oncl. 7: 625 (1996); Int. J. Cancer 73: 1021 (1997)), and organic cation transporter (Proc. Natl. Acad. Sci. USA 91: 133 (1994); Molec. Pharmacol. 51: 913 (1997)). Analysis of SNPs is being carried out for these drug-transporters in the same way as for drug-metabolizing enzymes. Transporter SNPs that bring about functional changes have been recently found. These SNPs are receiving attention as one of the factors causing fluctuations between individuals (Ryu S. et al., J. Biol. Chem. 275: 39617-39624 (2000); Tirona R. G. et al., J. Biol. Chem. 276: 35669-35675 (2001)). Currently, functional analysis of transporter SNPs mainly uses mammalian cells introduced with genes. However, this is speculated to be problematic for accurately detecting functional changes caused by SNPs in substrates having a low activity ratio compared to parent cell lines.